Covalent hybridization of CNT by thymine and uracil: A computational study

We have investigated the electronic and structural properties of covalent functionalization of the tip of (5,0) carbon nanotube (CNT) by di-keto and keto-enol forms of thymine (T) and uracil (U) nucleobases. Density functional theory (DFT) calculations have been performed to optimize the investigated structures and to calculate the properties such as dipole moment, bond length, band gap, total energy, binding energy and quadrupole coupling constant. The results indicated that, due to the functionalization of CNT by T and U, the hybrids exhibit new properties in which they are similar in both types of CNT-T and CNT-U hybrids.     

Carbon doped boron phosphide nanotubes: A computational study

A computational study based on density functional theory (DFT) calculations has been performed to investigate the properties of the electronic structure of carbon doped boron phosphide nanotube (C-doped BPNT). Pristine and the C-doped structures of two representative (6,0) zigzag and (4,4) armchair BPNTs have been investigated. At first, the geometries of the structures have been allowed to relax by optimization. Subsequently, NMR parameters have been calculated in the optimized structures. The results indicated that the influence of C-doping was more significant on the geometries of the zigzag model than the armchair one. The difference of band gap energies between the pristine and C-doped armchair BPNT was larger than the zigzag model. Significant differences of NMR parameters of those nuclei directly contributed to the C-doping atoms have been observed.     

Melatonin: A new inhibitor agent for cervical cancer treatment

Cervical cancer is one of the most common cancers between women and is known as the third leading cause of female cancer related deaths annually. Its detection in early stages allows it to be a preventable and generally treatable disease. Increasing evidence revealed, a variety of internal and external factors are associated with initiation and progression of cervical cancer pathogenesis. Human papilloma virus infection is found as a major cause of cervical cancer. Other molecular and biochemical alterations as well as genetic and epigenetic changes are related cervical cancer progression. Current treatment options often have severe side effects and toxicities thus, new adjuvant agents having synergistic effects and ability to decrease different side effects and toxicities are needed. Melatonin is an indolamine compound secreted from the pineal gland which shows wide range anticancer activities. A large amount of studies indicated inhibitory effects of melatonin against various types of cancers. In addition, experimental evidence reports inhibitory effects of melatonin as an adjuvant therapy on cervical cancer by targeting a sequence of different molecular mechanisms. Herein, for first time, we summarized anticervical cancer effects of melatonin and its underlying molecular mechanisms.     

A microscale anisotropic biaxial cell stretching device for applications in mechanobiology

A multi-layered polydimethylsiloxane microfluidic device with an integrated suspended membrane has been fabricated that allows dynamic and multi-axial mechanical deformation and simultaneous live-cell microscopy imaging. The transparent membrane’s strain field can be controlled independently along two orthogonal directions. Human foreskin fibroblasts were immobilized on the membrane’s surface and stretched along two orthogonal directions sequentially while performing live-cell imaging. Cyclic deformation of the cells induced a reversible reorientation perpendicular to the direction of the applied strain. Cells remained viable in the microdevice for several days. As opposed to existing microfluidic or macroscale stretching devices, this device can impose changing, anisotropic and time-varying strain fields in order to more closely mimic the complexities of strains occurring in vivo.     

Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics

Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases.Mass-spectrometry based proteomics provides various tools to detect and quantify changes in protein expression or post-translational modifications (PTMs).¹ In bottom-up proteomics, these analyses typically involve using peptides derived from the tryptic digestion of proteins. Although trypsin is a robust enzyme and provides peptides suitable for mass spectrometry, not all sequences are detectable by this approach (1). Sequences may be missed because of the limited number and uneven distribution of lysine and arginine residues throughout a protein sequence. Tryptic coverage of interesting regions of sequence, such as trans-membrane domains that may contain notable PTMs, is often incomplete (2). Sequence coverage greater than that offered by trypsin is a requirement for many studies (3).Missing sequence coverage can also adversely affect analysis by selected reaction monitoring (SRM). Although SRM has emerged in recent years as a highly sensitive and accurate method for protein detection and quantification (4), it is sometimes hampered by the limited number of targetable peptides (primarily tryptic peptides) available in public databases. Improving amino acid sequence coverage would provide more targets for SRM assay development, facilitating protein quantification and the ability to target specific isoforms or sequence regions of interest.Fractionation is commonly employed to increase protein identifications and improve sequence coverage, but introduces a number of complexities. Separation of proteins or peptides significantly increases the number of samples to analyze and the amount of data to process. Species may be present in multiple fractions or in different fractions in different runs, which makes quantitative analysis with techniques like SRM difficult. However, SRM has sufficient sensitivity that peptides identified in fractionated discovery experiments are often targetable in whole lysate (5).One approach to increase sequence coverage without fractionation or purification is to use proteases other than trypsin for digestion (6, 7). In recent years, there has been a surge in the use of alternative proteases to improve sequence coverage. Biringer et al. demonstrated in 2006 that combining the MS data from tryptic and Glu-C digestions of human cerebrospinal fluid (CSF) resulted in increased protein identifications. Sequence coverage also improved versus individual enzyme digests, though this was shown only for the 38 most confidently identified proteins (8). In 2010, Swaney et al. expanded the multi-enzyme approach to five specific proteases (trypsin, Lys-C, Arg-C, Asp-N, and Glu-C) and showed that although this method only modestly increases the number of protein IDs, it significantly increases the average sequence coverage (from 24.5% to 43.4%) (9). The most comprehensive coverage of a human cell line to date was reported by Nagaraj et al., in which in-depth proteomics with two levels of prefractionation and analysis using trypsin, Lys-C, and Glu-C was carried out for the HeLa cell line. A total of 10,255 proteins and 166,420 peptides were identified (10). However, none of these studies investigated the use of consecutive enzymatic digestion on a sample.The Mann laboratory recently introduced a strategy, using consecutive digestion in conjunction with filter-aided sample preparation (FASP), for two-step and three-step digestions with various combinations of trypsin, Lys-C, Glu-C, Arg-C, and Asp-N (11). The consecutive use of Lys-C and trypsin enabled the identification of up to 40% more proteins and phosphorylation sites in comparison to trypsin alone. However, a systematic study of all common commercially available proteases for comprehensive mapping of the human proteome has not yet been performed.These prior studies have clearly shown the ability of tandem and parallel protease digestion to improve protein ID and sequence coverage. However, their focus has been either to improve the number of protein identifications or to improve the sequence coverage of few targets. In an effort to provide a resource for targeting as much of the amino acid sequence in a human cell line as possible, we conducted a comprehensive study in which seven commercially available enzymes were used individually and in combination. First, we digested HeLa lysate with a total of 48 single, double, and triple enzyme combinations. Across these combinations we detected 5223 proteins with an average of 42% sequence coverage by analyzing the total cell lysate digest without fractionation. We then selected the best five complementary digests for each of Orbitrap elite collision induced dissociation (CID) and Q exactive higher-energy CID (HCD) analyses. A strong-anion exchange fractionation strategy was applied to these best digests, from which we were able to identify 8470 proteins with 40.3% mean sequence coverage. Combining all digests, both unfractionated and SAX, gave 8539 proteins with 44.7% mean coverage. These data are now publically available (https://proteomics.swmed.edu/confetti) and can be queried using a simple web interface to discover the enzyme or combination of enzymes required to yield a peptide spanning a certain region of interest on a protein.Finally, we performed a proof-of-concept experiment to demonstrate that SRM assays using nontryptic peptides are viable, and in some cases more sensitive than tryptic assays. Though tryptic peptides are generally sufficient for protein quantification by SRM we believe there will be increased use of nontryptic SRM as coverage of specific regions of sequence becomes more important. For example, bio-marker studies considering the presence of specific PTMs rather than general protein abundance are increasingly common. Truly comprehensive PTM studies require access to the nontryptic proteome.     

Protein-RNA cross-linking in the ribosomes of yeast under oxidative stress

Living systems have efficient degradative pathways for dealing with the fact that reactive oxygen species (ROS) derived from cellular metabolism and the environment oxidatively damage proteins and DNA. But aggregation and cross-linking can occur as well, leading to a series of problems including disruption of cellular regulation, mutations, and even cell death. The mechanism(s) by which protein aggregation occurs and the macromolecular species involved are poorly understood. In the study reported here, evidence is provided for a new type of aggregate between proteins and RNA in ribosomes. While studying the effect of oxidative stress induced in the yeast proteome it was noted that ribosomal proteins were widely oxidized. Eighty six percent of the proteins in yeast ribosomes were found to be carbonylated after stressing yeast cell cultures with hydrogen peroxide. Moreover, many of these proteins appeared to be cross-linked based on their coelution patterns during RPC separation. Since they were not in direct contact, it was not clear how this could occur unless it was through the RNA separating them in the ribosome. This was confirmed in a multiple-step process, the first being derivatization of all carbonylated proteins in cell lysates with biotin hydrazide through Schiff base formation. Following reduction of Schiff bases with sodium cyanoborohydride, biotinylated proteins were selected from cell lysates with avidin affinity chromatography. Oxidized proteins thus captured were then selected again using boronate affinity chromatography to capture vicinal diol-containing proteins. This would include proteins cross-linked to an RNA fragment containing a ribose residue with 2',3'-hydroxyl groups. Some glycoproteins would also be selected by this process. LC/MS/MS analyses of tryptic peptides derived from proteins captured by this process along with MASCOT searches resulted in the identification of 37 ribosomal proteins that appear to be cross-linked to RNA. Aggregation of proteins with ribosomal RNA has not been previously reported. The probable impact of this phenomenon cells is to diminish the protein synthesis capacity.     

Anti‐inflammatory and immune‐modulatory impacts of berberine on activation of autoreactive T cells in autoimmune inflammation

Autoreactive inflammatory CD4⁺ T cells, such as T helper (Th)1 and Th17 subtypes, have been found to associate with the pathogenesis of autoimmune disorders. On the other hand, CD4⁺ Foxp3⁺ T regulatory (Treg) cells are crucial for the immune tolerance and have a critical role in the suppression of the excessive immune and inflammatory response promoted by these Th cells. In contrast, dendritic cells (DCs) and macrophages are immune cells that through their inflammatory functions promote autoreactive T‐cell responses in autoimmune conditions. In recent years, there has been increasing attention to exploring effective immunomodulatory or anti‐inflammatory agents from the herbal collection of traditional medicine. Berberine, an isoquinoline alkaloid, is one of the main active ingredients extracted from medicinal herbs and has been shown to exert various biological and pharmacological effects that are suggested to be mainly attributed to its anti‐inflammatory and immunomodulatory properties. Several lines of experimental study have recently investigated the therapeutic potential of berberine for treating autoimmune conditions in animal models of human autoimmune diseases. Here, we aimed to seek mechanisms underlying immunomodulatory and anti‐inflammatory effects of berberine on autoreactive inflammatory responses in autoimmune conditions. Reported data reveal that berberine can directly suppress functions and differentiation of pro‐inflammatory Th1 and Th17 cells, and indirectly decrease Th cell‐mediated inflammation through modulating or suppressing other cells assisting autoreactive inflammation, such as Tregs, DCs and macrophages.     

Neuroprotective Effects of Methyl-3-O-methyl gallate Against Sodium Fluoride-Induced Oxidative Stress in the Brain of Rats

Methyl-3-O-methyl gallate (M3OMG) is a rare natural product that showed promising in vitro antioxidant activities. In this study, the protective role of synthetic M3OMG against sodium fluoride (NaF)-induced oxidative stress in rat brain was evaluated. Animals were treated with either M3OMG (10 and 20 mg/kg i.p.), vitamin C (10 mg/kg i.p.) as the standard antioxidant or the vehicle (5 % dimethyl sulfoxide; 1 ml/kg) for 1 week. Oxidative stress was induced in the brain by adding 600 ppm NaF in the drinking water for 7 days. At the end of the treatment period, the levels of thiobarbituric acid reactive substances (TBARS), reduced glutathione and the activities of antioxidant enzymes (superoxide dismutase and catalase) were evaluated in brain homogenates. M3OMG treatment mitigated the NaF-induced oxidative stress through normalization of the level of TBARS, reduced levels of glutathione and by the restoration of the diminished antioxidant enzyme activities. In conclusion, M3OMG could have a potential for treating neurotoxicity induced by fluoride or related environmental pollutants.     

ROS-mediated heme degradation and cytotoxicity induced by iron nanoparticles: hemoglobin and lymphocyte cells as targets

Nanoparticles (NPs) due to their small size and high surface area induce remarkable adverse effects on the biological systems. However, the exact mechanism by which NPs interacted with biological system and induce their adverse effects is still an enigma. Herein, the interaction of zero valent iron NPs (ZVFe NPs) with human hemoglobin (Hb) was evaluated using a variety of techniques including circular dichroism, fluorescence, and UV–visible (UV–vis) spectroscopy methods. Also, the cytotoxicity of ZVFe NPs on the human lymphocyte cell line as a model of blood system cell line was investigated by reactive oxygen species (ROS), caspase-9, and caspase-3 activities assays. It was revealed that ZVFe NP interaction resulted in heme displacement and degradation and induction of protein cabonylation. It was also shown that ZVFe NPs impaired the complexity of lymphocyte cells through ROS generation and apoptotic pathway. Together, these data suggest that NPs influence the biological system and induce adverse effects through ROS generation.